[Purpose] Different six types of assisted hatching (AHA) were applied to mouse embryo and their effect on hatching was investigated. [Methods] The zona pellucida was treated as follows. (a) Piercingness (PIC): Pierce the zona with a very fine needle. (b) Partial zona dissection-small (PZD-small): Proceed the same as with PZD in microinsemination and make an incision about 1/8 the circumference in the zona. (c) Partial zona dis-section-large (PZD-large): Enlarge the incision about 1/4 the circumference. (d) Zona drilling (ZD): Spray acid solution to the zona, which dissolves the zona to create a hole about 30 μm in diameter. (e) Zona thinning-par-tial (ZT-partial): Thin down a part of the outer area of the zona to about half the thickness by the same procedure as in ZD. (f) Zona thinning-total (ZT-total): Thin down the total outer area of the zona. [Results] When these AHA were each administered to 4-cell-stage embryos, the effects of PZD-large, ZD, ZT-partial and ZT-total on the hatching were observed, but the effects of PIC and PZD-small were not recognized. Furthermore the effects of AHA on 8-cell-stage embryo, morula and frozen-thawed embryo were recognized. With PZD-partial in which the opening of incision is small, the embryo in the halfway through hatching was trapped in the thick zona pellucida. With PZD-large or ZD in which an incision or a hole is large, hatching started at the earlier stage than normal (premature hatching). [Conclusion] With the role of the zona and the time to start hatching taken into consideration, zona thinning (ZT-partial or ZT-total) appears to become the most reasonable AHA.